The lymphatic vascular system is essential for fluid balance, fat absorption, and coordination of the immune response. Lymphatic vessels in mammals arise from pre-existing blood vessels to form a separate vascular network but the molecular signals that direct the establishment and maintenance of the lymphatic system are mostly unknown. SLP-76 is an intracellular adaptor protein in the immunoreceptor signaling pathway that lies downstream of the tyrosine kinase SYK and upstream of the phospholipase PLCg2. In mice loss of SYK, SLP-76, or PLCg2 results in embryonic hemorrhage and perinatal death, phenotypes that cannot be explained entirely by associated platelet-signaling defects. We have identified failure to separate lymphatic and blood vessels during lymphatic vessel formation as the major cause of the hemorrhagic phenotype in SLP-76-deficient animals. Abnormal fusion between blood and lymphatic vessels during development leads to hemorrhage from leaky, blood-filled lymphatics and the formation of arterio-venous shunts, which result in an elevated cardiac output in surviving mice. Our findings suggest that SLP-76 participates in a novel angiogenic signaling pathway required for separation of vascular networks. To date, SLP-76 function has only been described in hematopoietic cells and not in any previously characterized angiogenic signaling pathways. The goal of this proposal is to determine how SLP-76 signaling regulates separation of blood and lymphatic vessels during angiogenesis. We hypothesize that SLP-76 is required directly or indirectly for the generation of repulsive signals between blood and lymphatic endothelial cells during lymphatic vessel development. Our aims are: 1. To define lymphatic development in mice lacking SLP-76 and its signaling partners SYK and PLCg2. 2. To identify SLP-76 domains essential for angiogenesis. 3. To determine if the requirement for SLP-76 during lymphatic development is cell autonomous. 4. To test for loss of repulsion between SLP-76-deficient blood and lymphatic endothelial cells. These studies will define a novel angiogenic signaling pathway required for lymphatic development and the separation of distinct vascular networks. [unreadable] [unreadable]